This project is designed to provide an in-depth understanding of the beta oxidation of unsaturated fatty adds, including polyunsaturated fatty adds. The focus of this study are the reactions and auxiliary enzymes which are specific to the beta-oxidation of unsaturated fatty acids. Most important is an investigation of the beta oxidation of unsaturated fatty adds with odd-numbered double bonds by the pathway involving delta 3,5, delta 2,4-dienoyl-CoA isomerase and 2,4-dienoyl CoA reductase. The contribution of this pathway to the mitochondrial beta-oxidation of 5- enoyl.CoA intermediates formed from linolenic add and oleic add will be determined. To facilitate such study with whole mitochondria, a mechanism-based inhibitor of delta 3,5, delta 2,4-dienoyl-CoA isomerase will be developed. The operation of this pathway in peroxisomes will be studied by determining the presence of delta 3,5, delta 2,4,-dienoyl-CoA isomerase in this organelle. The ongoing investigation of 2,4-dienoly-CoA reductase will be continued by cloning, sequencing and overexpressing the reductase gene from E. coli. The availability of a sufficent amount of the reductase will permit a study of its reaction mechanism. The substrate specificity of the peroxisomal reductase will be explored to aid in its purification. The notion that 3-hydroxyacyl-CoA epimerase may function in the metabolism of D-3-hydroxymyristic acid in E. coli will be investigated by isolating and characterizing epimerase mutants. The possible function of rat liver D-3-hydroxyacyl-CoA dehydratase, a peroxisomal enzyme essential for epimerase activity, in saturated fatty acid oxidation will be assessed by investigating its copurification with a suspected D-3-hydroxyacyl-CoA dehydrogenase. Finally the beta-oxidation of cis and trans unsaturated fatty acids in mitochondria will be compared with the aim of detecting and analyzing differences between their rates of degradation. This project will significantly increase the current knowledge of unsaturated fatty add beta-oxidation and thereby aid in the diagnosis of inherited diseases of fatty add oxidation and in assessing the metabolic consequences of trans fatty acid ingestion.